Device and method for detecting bioelectric signals from electrophysiologically active regions in spheroids

ABSTRACT

Disclosed is a device for detecting bioelectric signals from spheroids comprising a measuring chamber having a measuring chamber wall which encloses a volume, which is open at least at one side, is composed of an electrically non-conducting material, and has, in at least one measuring region, an inner cross section, which corresponds as far as possible to the largest cross section of a spheroid, comprising at least a number of electrodes which are disposed in a common plane inside said measuring chamber wall and each electrode has a freely accessible electrode surface which is oriented towards the measuring region, and comprising an impedance measuring arrangement which is connected to the electrodes. The device and the method can be used to test substances in 3D biological in-vitro (three-dimensional) cell aggregates.

TECHNICAL FIELD

The present invention relates to a device and a method for detectingbioeletric signals from electrophysiologically active regions inspheroids. In particular, it is described how the effect ofpharmaceutical preferably neuropharmacological or neurotoxic substancescan be detected without damaging the spheroids so that the spheroidscontinue to be at disposal for further study possibilities.

STATE OF THE ART

In order to be able to routinely determine the effect of substances, forexample pharmacological substances, on living systems, in recent yearsbiosensors have been developed, which are based on living cells, seeBousse, L., “Whole Cell Biosensors”, Sensors and Actuators, volume 34,pp., 270-275 (1996). Such type biosensors that are based on biologicalcells are primarily provided with mono-layer cell cultures as abiological detection system, but substance-caused complex cell/cell orcell/matrix interactions can often not be determined with the desiredprecision and reliability. Furthermore, the effect ofneuropharmaceuticals or environmental toxins indeed leads to thesecomplex cell/cell interactions in the central nervous system, which needto be ascertained in order to obtain further insight into thebiochemical reaction chain of such substances on biological cellmaterial. Finally, the biosensors based on mono-layer cell cultures havethe drawback that the measuring results obtained using them only providelimited information about the actual reaction capabilities of thebiological cells, for example to selective application of a substance,because the mono-layer cell cultures do not exist in this from in livingnature.

In order to avoid this drawback, biological models approximating an invivo situation with regard to the intercellular as well as intracellularinteractions as closely as possible must be resorted to when studyingsuch type substances. Three-dimensional cell systems reflect an in vivosituation substantially better than single cells or mono-layer cellcultures. Therefore, it is necessary to use three-dimensional cellsystems to test substances which are intended for influencing cell/cellinteractions.

In order to test the neuropharmacological or neurotoxic effect ofsubstances, for example beyond animal models, bioelectric signals aredetermined in a prior art manner from the ex vivo tissue sections withthe aid of glass micro-electrodes or needle electrodes. Planar electrodearrangements, so-called multi-electrode arrays are utilized to recordthe signal courses using multi-channel derivations. However, ex vivotissue sections must be prepared in a very complicated manner fromanimal models, cannot be standardized and are limited to the existentanimals models. Moreover, as ex vivo tissue sections degenerate rapidly,tissue sections are not suited for long-term testing. Long-term testing,however, is of extreme relevance for testing neuropharmaceuticals orenvironmental toxins and their influence on biological tissue.

An interesting research object for the preceding problem are so-calledspheroids, which may be considered as bead-shaped cell aggregates. Fromliterature are known, for example, research in retina genesis and retinaregeneration in which such type regenerated bead-shaped cell aggregates,so-called retino-spheroids, are obtained under constant conditions (seeMoscona, A. A, “Development of Heterotypic Combination of DissociatedEmbryonic Chick Cells”, Proc. Soc. Exp. Bio. Med 292, pp. 410-416(1956); Vollmer, G. Layer, P. G., Gierer, A.: “Reaggreation of EmbryonicChick Retina Cells: Pigment Epithelial Cells Induce a High Order ofStratification”, Neurosci. Lett. 48, pp. 191-196 (1984)). Theseregenerated bead-shaped cell aggregates are reaggregated by suitedcultivation of dissociated cells from embryonic retinae.

DE 199 46 458.8 describes a device and a method for characterizingspheroids by means of impedance spectroscopy. The influence ofsubstances on the proliferation, morphology and membrane properties ofthe in vitro tissue, i.e. outside the living organism, can be determinedwith this device and method. Locally resolved information from insidethe spheroid can, however, not be obtained with this prior-art method.Moreover, information about the intracellular electric potentials in theform of so-called bioelectric signals, from which the effect ofpharmaceutical substances, in particular neuro-pharmaceutical orneurotoxic substances can be determined, cannot be obtained with thedevice described in the preceding printed publication.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a device and a methodfor detecting bioelectric signals from spheroids in such a manner thatit is possible to determine the neurotoxic and neuropharmacologicaleffect of substances on biological tissue by way of in-vitro study asclose as possible to the in-vivo situation with regard to intercellularand intracellular interactions.

The solution of the object on which the present invention is based isset forth in claim 1. The subject matter of claim 14 is an inventivemethod. Features that advantageously develop the inventive idea are thesubject matter of the subclaims and are given in the description of theinvention with reference to preferred embodiments.

A key element of the present invention is that the device for detectingbioelectric signals from spheroids comprises the following components:

-   -   a measuring chamber having a measuring chamber wall, which        encloses a volume that is open at least on one side, is made of        non-electroconductive material and has an inner cross section at        least in one measuring region, which corresponds maximally to        the largest cross section of a spheroid.

The measuring chamber is preferably designed as a capillary withcapillary walls as well as a capillary bottom, which define themeasuring region of the spheroid. The size of the cross section of themeasuring region enclosed by the capillary walls is selected in such amanner that the spheroid is in mechanical contact with the measuringchamber respectively capillary wall along the spheroid's biggestcircumferential edge in such a manner that the spheroid assumes a fixedas possible spatial position inside the measuring region, which is ofgreat advantage for further measurement of the spheroid. In order tofurther improve the positioning of the spheroid inside the measuringchamber, respectively the capillary, in a preferred embodiment, thedevice is connected in the capillary bottom to a partial vacuum conduitto affix the spheroid inside the measuring region by means of suction.

-   -   at least a number of electrodes which are disposed in a common        plane inside the wall of the measuring chamber, the electrodes        each having one freely accessible electrode surface oriented        towards the measuring region. The electrodes are preferably        disposed in that plane within the wall of the measuring chamber,        in which the spheroid comes in contact with the measuring        chamber wall with the edge of its greatest circumference. The        requirement that the electrodes are disposed in a plane is not        necessarily to be understood as mathematically exact, i.e. in        the sense of along an imaginary line running around the inner        wall of the measuring chamber. The electrodes should be        disposed, at least with the surfaces oriented towards the        measuring region, along the region of contact between the        spheroid and the wall of the measuring chamber in such a manner        that the impedance distribution can be detected locally resolved        in the cutting plane inside the spheroid predetermined by the        electrode configuration with an impedance measuring arrangement        that is connected to the individual electrodes. For this        purpose, an electric current is induced inside the spheroid via        the single electrodes and the diminishing electric voltage over        the spheroid is measured. The impedance is formed from the        current and the voltage. In order to conduct so-called impedance        imaging, the frequency of the current induced in the spheroid is        varied over a continuous frequency range, and the impedance        yielded as a function of the frequency is recorded. Thus, with        the aid of such an impedance imaging system, the tissue        parameters can be determined locally resolved inside the cutting        plane from the recorded impedance distribution at different        frequencies. In this manner, information is gained about the        internal structure of the spheroid inside the cutting plane. For        instance, so-called electrophysiologically active regions, which        may differ in impedance behavior from the other not organized        regions inside the spheroid, are distinguished by a subunit with        a compact consistency. Especially these electrophysiologically        active regions are of particular interest in discovering how        certain substances influence biological cells, because it is in        these regions that scientifically detectable and evaluatable        signals are generated as a sort of cell response to the        substance's effect on the respective cell.        Electrophysiologically active regions inside the spheroid        possess a bioelectrical activity which influences the behavior        of the electrical surface potential of the entire spheroid. When        there is a change in the bioelectrical activity of the        electrophysiological active regions, for example, due to the        effect of a certain substance on the spheroid and, therefore,        simultaneously on the electrophysiologically active regions,        this directly influences the surface potential of the spheroid.        Preferably with the aid of a potential determining system, which        is connected to the electrodes disposed around the spheroid, it        is possible to detect the surface potentials along the cutting        plane through the spheroid and to obtain information about the        bioelectrical activity of the electrophysiologically active        regions inside the cutting plane.

For both impedance measurement and detection of the surface potentials,the free electrode surfaces do not necessarily have to be in directcontact with the surface of the spheroid.

But rather a culture fluid, for example representing a nutrient insidewhich the spheroid is generated, introduced into the measuring chamberalso acts as an electrically conducting medium through which anelectrical contact can be produced between the electrodes and thesurface of the spheroid.

In a simple embodiment, the free electrode surfaces-connect flush withthe inner wall of the measuring chamber in such a manner that a directcontact between the electrode surfaces and the spheroid prevails.

In an alternative embodiment, the electrodes are located in such amanner inside the so-called connecting chambers, which open on one sideinto the measuring chamber, that the free electrode surfaces are setback from the inner wall of the measuring chamber. The advantage of thisis first that the electrodes are easier to exchange respectivelyreplace. Moreover, with suited design and arrangement of the connectingchamber, for example, tapering conically towards the measuring chamber,larger free electrode surfaces can be utilized. With regard to a smallas possible phase limit impedance, the use of as large as possibleelectrode surfaces is desirable, which can be realized by correspondingspaced placement of the inner wall of the measuring chamber inside theconically designed connecting chambers. As already mentioned in thepreceding, the culture fluid, which is introduced into the measuringchamber together with the spheroid, acts as an electrical contact mediumbetween the electrodes and the spheroid surface.

With regard, in particular, to studying spheroids in industrial amountsto test how new pharmacological substances act, semiconductor materialsare suited for-setting up the device described in the preceding. Amultiplicity of array-like arranged measuring chambers, which areadapted in shape and size to studying spheroids and thus permitstatistical evaluation due to the great number of examined spheroids,can be realized with the aid of semiconductor technology. A concreteembodiment of this is described in more detail further on with referenceto the figures.

With the aid of the preceding device, the spheroids can be studied fortheir bioelectrical activity without destroying them, to then returnthem safely to a culture medium for further observation. Thus, one andthe same spheroid can be measured several times at intervals in order tobe able to determine possible signs of substance-caused degradation. Inthis manner, conclusions can be drawn statistically about how substancesact following evaluation of a multiplicity of such spheroids which areadditionally exposed to a certain substance inside a culture medium.

The invented method for detecting bioelectric signals from spheroids isdistinguished in particular by the combination of the following methodsteps: provision of a device of the type described in the preceding,placement and positioning of a spheroid inside the measuring chamber,and conducting an impedance measurement according to the impedanceimaging method for locally resolved determination ofelectrophysiologically active regions in the spheroid. In order to beable to determine any bioelectrical activity, an additional surfacepotential determination is conducted along the cutting planepredetermined by the configuration of the electrodes.

With the aid of the invented method, the morphology of themulti-cellular spheroids can be determined locally resolved in anon-invasive manner and, moreover, the excitation courses of theelectrophysiologically active regions can be precisely determined. Themethod permits, in particular, to be able to non-invasively detect theeffect of substances respectively drugs on 3D in vitro models of thecentral nervous system. The device, in the sense of a biosensor systemdescribed in the preceding, permits the realization of long-term studiesof neurotoxic and neuropharmacological effects of substances. Thespheroid utilized as a biological detection element is only positionedin the measuring chamber for a short period during impedance measurementand potential determination and can, independent of the measuringarrangement, be cultivated under physiological conditions. Adhesion ofthe spheroid is largely prevented by the presence of the culture fluidinside the measuring chamber and undesired cell/material interactionsare minimized. Depending on the question to be resolved, spheroids or 3Dbiological detecting elements can be generated with different types ofcells in different positions for the biosensor system.

BRIEF DESCRIPTION OF THE DRAWING

The present invention is made more apparent in the following, withoutthe intention of limiting the scope or spirit of the overall inventiveidea, using preferred embodiments with reference to the accompanyingdrawings. Shown is in:

FIG. 1 a schematic flow chart representation of carrying out the methodof analysis,

FIGS. 2 a,b representation of a measuring chamber with a spheroid,

FIGS. 3 a,b,c representations of cross sections of a spheroid andan-image of the potential,

FIG. 4 a cross section of an array-shaped measuring arrangement insemiconductor technology,

FIG. 5 measuring arrangement

FIG. 6 a cross section of a measuring chamber and

FIG. 7 a cross section of an alternative measuring chamber.

WAYS TO CARRY OUT THE INVENTION, COMMERCIAL APPLICABILITY

The invented method is explained with reference to FIG. 1 using a studyof reaggregated retino-spheroids as an example:

Under micro-gravitation conditions in a bioreactor 1, dissociatedembryonic cells of the central nervous system are reaggregated tobead-shaped neuronal reaggregation cultures, the so-calledretinospheroids. With the addition suited growth factors and/or suitedgenetic manipulations, it is achieved that electrophysiologically activecell regions form evenly distributed in the spheroid. Thus with highprobability, at least one electrophysiologically active region islocated in a random cutting plane running through the center of thespheroid.

In order to test the effect of a substance on the spheroid, at least onespheroid 2 has to be isolated from the bioreactor 1 and placed in themeasuring chamber of the biosensor system 3 to test it there as an invitro model. In the bioreactor 1 as well as in the measuring chamber ofthe biosensor 3, the spheroid 2 is located in a culture fluid,respectively in an analyt, so that the spheroid is replaceable asdesired without impairment between the bioreactor and the measuringchamber.

By means of multi-frequency impedance imaging 4, the position and theextension of the various cell regions is determined in a cross sectionplane predetermined by the electrode configuration inside the measuringchamber. And then the bioelectrical activity of the individual cellregions in the cutting plane is detected by means of the electricalsource-imaging 5. Systems and algorithms for the impedance imaging andthe electric source imaging are fundamentally known from medicaltomography, see Webster, J. G., “Electrical Impedance Tomography”, AdamHilger, Bristol (1990).

Changes in the electrophsiological activity of certain regions in thespheroid, correlation of the electrophysiological activity of differentregions and the change in the tissue parameters serve as parameters 6for the effect of substances on the in vitro tissue model.

To conduct the impedance imaging and the potential determination, anelectrophysiologically active spheroid 2 according to FIG. 2 ispositioned in the desired culture stage in a measuring chamber 7.Depending on the question to be resolved, such as for example long-termstudies or dynamic excitation, the to-be-tested substance is added tothe culture fluid in the bioreactor or to the culture fluid in themeasuring chamber 7. The measuring chamber 7 is preferably formed by acapillary 8, which is designed cylindrical in the positioning region andits wall 9 is made of an electrically insulated material. In thepositioning region of the capillary 8, a multiplicity of electrodes 10are disposed in the capillary wall 9 in at least one plane perpendicularto the longitudinal axis. As the electrodes 10 with their free electrodesurfaces in the preferred embodiment shown in FIGS. 2 a,b are placedflush with the inside wall of the measuring chamber, they come incontact with the spheroid (2) placed inside the measuring chamber in acutting plane along the spheroid's greatest circumference (FIG. 2 b).The electrodes are electrically contacted singly from the outside fortriggering (not depicted).

This arrangement is used both for locally resolved determination of thepassive electrical properties of the in-vitro tissue and fordetermination of the spatial and temporal course of theelectrophysiological excitation.

In order to determine the impedance distribution in the cutting plane ofthe spheroid in which the electrodes lie, the electrodes are connectedin a suited manner with an impedance imaging system. The tissueparameters are determined locally resolved from the impedancedistributions at different frequencies. FIG. 3 a shows an actual sectionthrough a spheroid with the usually not further organized regions 11,organized subregions, the so-called electrophysiologically activeregions 12 and inner fiber layers 13. FIG. 3 b shows a section imagedetermined by means of impedance imaging, which really corresponds tothe section image shown in FIG. 3 a. The bioelectrical activity ofcertain regions is determined from the surface potentials, which aredetermined with the electrodes, and from the impedance distribution, seediagrammatic representation in FIG. 3 c. In the determination of thesurface potentials, the electrodes of the measuring capillaries areconnected to a measurement system. Marked evaluatable measured signalsare, in particular, the voltages peaks perceivable on the surface of thespheroid (see diagrammatic representation) and their temporal sequence(Δt₁, Δt₂). In particular, these measured values are the ones that areprovenly influenced by the presence of certain substances, which allowsdrawing conclusions about the effect of certain substance on biologicalmaterial.

FIG. 4 shows a measuring chamber arrangement in which a multiplicity ofsingle measuring chambers 7 is disposed in an array structure on aplanar substrate 14. In order to produce such a measuring chamberarrangement, a silicon nitride layer 15 with a thickness ofapproximately 1 μm is deposited on a silicon substrate 14. The siliconnitride layer 15 is exposed from the underside as membranes.Furthermore, microholes 16 with a diameter of 20 μm are produced in themembrane 15 by means of dry etching. Finally, a phototresist 17 with athickness of 40 μm is applied. Cylindrically shaped measuring chambers 7(diameter of 150 μm) are etched free into the photoresist 17 concentricto the microholes. A metal layer with a thickness of 10 μm is depositedonto the photoresist 17 and structured in such a manner that themeasuring chambers 7 are surrounded by eight electrodes 10 disposedevenly spaced in a circle. Finally another photoresist layer 17 (50 μm)is deposited and structured.

In order not to damage the spheroids when placing them into theindividual measuring chambers 7, the edges of the measuring chambers 7are rounded off. In order to be able to apply a partial vacuum 18 forpositioning the spheroids, the finished microstructure is glued onto aplate with a borehole and tube connection. To conduct a measurement, theentire region of the measuring chamber 7 is filled with culture fluid 19to prevent adhesion effects between the individual spheroids and themeasuring chamber wall.

The electrodes 10 placed in the measuring chamber, according to FIG. 5,are connected with an impedance measuring system 21 and a potentialdetermination system 22 via a multiplexer 20. The measured data aretransmitted to a data collection and data analysis unit 23, which alsocontrols the multiplexer 20.

According to the preferred embodiment in FIG. 6 showing a cross sectionof a measuring chamber 7, connecting chambers 24, which end in themeasuring chamber, are disposed in a star-shaped manner runningconically into the measuring chamber 7. In this way, the size of themetal electrodes 10, which are each contacted by strip conductors 10*,can be selected independent of the size of the measuring chamber, andthe phase limit impedance of the electrode 10 can be reduced by largerelectrode surfaces.

In another preferred embodiment according to FIG. 7, in order to realizeimpedance measurements, one electrode 10′ for supplying current and oneelectrode 10″ for determining the potential are disposed in each channelin a four-electrode configuration.

LIST OF REFERENCE NUMBERS

-   1 bioreactor-   2 spheroid-   3 biosensor, measuring arrangement-   4 impedance measuring arrangement-   5 potential determining arrangement-   6 evaluation parameter-   7 measuring chamber-   8 capillary-   9 measuring chamber wall-   10 electrode-   10* strip conductor-   10′ current supply electrode-   10″ potential determining electrode-   11 not organized region-   12 organized region, electrophysiological region-   13 inner fiber layer-   14 substrate-   15 membrane-   16 microhole-   17 photoresist-   18 vacuum connection-   19 culture fluid-   20 multiplexer-   21 impedance system-   22 potential determining system-   23 data collection and analysis system-   24 connecting chamber

1. A device for detecting bioelectric signals from spheroids comprisinga measuring chamber that is pot-shaped, said measuring chamber having ameasuring chamber wall which encloses a volume which is open at oneside, wherein the measuring chamber is composed of an electricallyinsulating material and has, in at least one measuring region, an innercross section which corresponds to the largest cross section of aspheroid and is sized such that the spheroid is in contact with themeasuring chamber wall, at least a number of electrodes which aredisposed in a common plane inside said measuring chamber wall and eachsaid electrode has a freely accessible electrode surface which isoriented towards said measuring region, and an impedance measuringarrangement which is connected to said electrodes.
 2. The deviceaccording to claim 1, wherein a potential determining system isconnected to said electrodes.
 3. The device according to claim 2,wherein provided in said measuring chamber wall is a number ofconnecting chambers which are openly connected with said measuringregion and are disposed in a common plane evenly distributed around saidmeasuring region in circumferential direction, and inside each of saidconnecting chamber an electrode is placed, whose said freely accessibleelectrode surface oriented towards said measuring region is spaced at adistance from said measuring chamber inner wall.
 4. The device accordingto claim 3, wherein a second electrode is arranged inside each of saidconnecting chambers and being connected to said potential determiningsystem.
 5. The device according to claim 2, wherein said impedancemeasuring arrangement and said potential determining system areconnected to said electrodes via a multiplexer.
 6. The device accordingto claim 2, wherein a data collecting and data evaluation unit isconnected to said impedance measuring arrangement as well as to saidpotential determining system.
 7. The device according to claim 1,wherein said measuring chamber is designed as a cylindrical capillary,and said at least a number of said electrodes is disposed in one planeorthogonally to the length of said capillary.
 8. The device according toclaim 1, wherein said freely accessible electrode surfaces of saidelectrodes are designed flush with said measuring chamber inner wall. 9.The device according to claim 1, wherein said measuring chamber can befilled with an electrically conducting liquid.
 10. The device accordingto claim 1, wherein a partial vacuum conduit is connected to saidmeasuring chamber.
 11. The device according to claim 1, wherein apartial vacuum conduit is provided at the bottom of said pot forpositioning and attaching a spheroid placed in said pot-shaped measuringchamber by means of a partial vacuum.
 12. The device according to claim1, wherein a multiplicity of measuring chamber is arranged in anarray-like manner and is designed in planar semiconductor substratetechnology.
 13. The device according to claim 1, wherein said electrodesare disposed evenly distributed in circumferential direction of saidmeasuring chamber wall.
 14. A method for detecting bioelectric signalsfrom spheroids through a device that includes a measuring chamber thatis pot-shaped, said measuring chamber having a measuring chamber wallwhich encloses a volume which is open at one side, wherein the measuringchamber is composed of an electrically insulating material and has, inat least one measuring region, an inner cross section which correspondsto the largest cross section of a spheroid and is sized such that thespheroid is in contact with the measuring chamber wall, at least anumber of electrodes which are disposed in a common plane inside saidmeasuring chamber wall and each said electrode has a freely accessibleelectrode surface which is oriented towards said measuring region, andan impedance measuring arrangement which is connected to saidelectrodes, the method comprising: placing and positioning a spheroidinside said measuring chamber, and conducting an impedance measurementbased on a locally resolved determination of electrophysiologicallyactive regions in said spheroids.
 15. The method according to claim 14,wherein a surface potential determination is conducted for detectingsaid bioelectrical activity.
 16. The method according to claim 14,wherein said impedance measurement is conducted at different triggeringfrequencies to obtain an impedance spectrum.
 17. The method of claim 14,wherein the measuring chamber includes a culture fluid, and the freeelectrode surfaces are placed flush with the measuring chamber wall, themethod comprising: individually contacting each electrode from outsidethe measuring chamber.
 18. A method for detecting bioelectric signalsfrom spheroids through a device that includes a measuring chamber thatis pot-shaped, said measuring chamber having a measuring chamber wallwhich encloses a volume which is open at one side, wherein the measuringchamber is composed of an electrically insulating material and has, inat least one measuring region, an inner cross section which correspondsto the largest cross section of a spheroid and is sized such that thespheroid is in contact with the measuring chamber wall, at least anumber of electrodes which are disposed in a common plane inside saidmeasuring chamber wall and each said electrode has a freely accessibleelectrode surface which is oriented towards said measuring region, andan impedance measuring arrangement which is connected to saidelectrodes, the method comprising: removing a spheroid to which asubstance has been applied from a culture medium and placing saidspheroid in said measuring chamber of said device; performing at leastone of an impedance measurement and a potential determinationnon-invasively on said spheroid; and returning said spheroid unharmed tosaid culture medium.
 19. The method according to claim 18, wherein saidsubstances are pharmaceutical substances, in particular,neuropharmacological or neurotoxic substances.